Acute lymphoblastic leukemia (ALL) is the most common form of cancer in children (Pizzo and Poplack, 1993, Greaves, 1986, Uckun et al., 1998, Crist et al., 1988). A better understanding of the biological basis and predisposing leukemogenic events in this disease is needed in order to develop more effective treatment programs as well as novel prevention strategies.
Leukemic clones are thought to originate in ALL patients from normal lymphocyte precursors arrested at various stages of T- or B-lymphocyte development (Greaves, 1986). Accordingly, any critical regulatory network that controls normal lymphocyte development is a potential target for a leukemogenic event.
One such regulatory network vital for normal hematopoiesis involves Ikaros, a member of the Kruppel family “zinc finger” DNA-binding proteins. Ikaros acts as an evolutionarily conserved “master switch” of hematopoiesis that dictates the transcriptional regulation of lymphocyte ontogeny and differentiation (Georgopoulos et al., 1994, Georgopoulos et al., 1992, Hahm et al., 1994, Molnar and Georgopoulos, 1994, Wang et al., 1996, Winandy et al., 1995, Molnar et al., 1996, Sun et al., 1996, Hansen et al., 1997, Georgopoulos et al., 1997, Brown et al., 1997, Klug et al., 1998).
The programmed expression and function of the Ikaros gene is tightly controlled by alternative splicing of the Ikaros pre-mRNA which results in production of eight different Ikaros isoforms. All eight Ikaros isoforms share a common carboxy(C)-terminal domain containing a transcription activation motif and two zinc finger motifs that are required for hetero- and homodimerization among the Ikaros isoforms and for interactions with other proteins (Hahm et al., 1994, Molnar and Georgopoulos, 1994, Sun et al., 1996). Only three of the eight Ikaros isoforms, however, contain the requisite three or more amino(N)-terminal zinc fingers that confer high affinity binding to an Ikaros-specific core DNA sequence motif in the promoters of target genes (Sun et al., 1996).
The formation of homo- and heterodimers among the DNA binding isoforms increases their affinity for DNA, whereas heterodimers between the DNA binding isoforms and non-DNA binding isoforms are unable to bind DNA. Therefore, Ikaros proteins with fewer than three N-terminal zinc fingers exert a dominant negative effect by interfering with the activity of Ikaros isoforms that can bind DNA (Molnar et al., 1996, Sun et al., 1996). Thus, splicing errors can have severe consequences for the lymphocyte compartment of the developing immune system. An abundance of dominant-negative Ikaros isoforms that no longer bind DNA could result in significantly impaired expression of regulatory target genes that are essential for the orderly development and maturation of lymphocyte precursors.
In mice, absence of the normal Ikaros gene results in an early and complete arrest in the development of all lymphoid lineages during both fetal and adult hematopoiesis (Georgopoulos et al., 1994). Ikaros-deficient mice have a rudimentary thymus, lack peripheral lymph nodes, and are characterized by a complete absence of lymphocyte progenitor cells as well as mature B-lymphocytes, T-lymphocytes, and natural killer cells (Georgopoulos et al., 1994). Mice heterozygous for a germline mutation which results in the loss of critical DNA-binding zinc fingers of Ikaros develop a very aggressive form of lymphoblastic leukemia with a concomitant loss of the single wild type Ikaros allele between three and six months after birth (Winandy et al., 1995). Finally, the most recent findings in ALL molecular etiology show a pivotal role for Ikaros gene regulation in lymphoblast neoplastic transformation in infants (Sun et al., 1999) with T-lineage or B-lineage ALL leukemic cells expressing high levels of dominant-negative Ikaros isoforms.
It has long been suspected that molecular rearrangements in the lymphoid lineage precursors leading to ALL occur during fetal hematopoiesis (Ford et al., 1993, Gill Super et al., 1994). With the prospect of Ikaros malfunction and Ikaros isoform expression being at the core of leukemogenesis, a better understanding of the events taking place during embryonic blood cell differentiation is required in order to develop rational therapies. To address this need, an adequate experimental model system of vertebrate hematopoiesis is essential.
The zebrafish (ZF), with its extremely rapid embryonic development (3 days) and short maturation period (2-3 months) offers an attractive model. Over the past decade, the ZF embryo has been used to study eukaryotic gene activity and intercellular signaling in vertebrate development (Nusslein-Volhard, 1994, Zhang et al., 1998, Nguyen et al., 1998), and has emerged as a powerful genetic system, strongly relevant to the study of molecular medicine (Driever and Fishman, 1996, Amemiya, 1998). Intensive study of early embryonic hematopoiesis in the ZF along with the generation of hematopoietic mutants has turned the ZF into a useful model for the study of human blood disorders, such as congenital sideroblastic anemia (Brownlie et al., 1998) and hepatoerythropoietic porphyria (Wang et al., 1998). (See detailed reviews: Bahary and Zon, 1998, Amatruda and Zon, 1999).
It has now been discovered that transient, inappropriate expression during early embryonic development of the non-DNA binding Ikaros forms, including the dominant-negative isoforms, mutant forms, and others, have a significant impact on blood cell differentiation at later stages of development. Using the transgenic animal model of the invention, the effect of various agents on blood cell differentiation can be efficiently assessed. The ZF, with its relatively large and translucent embryo, external fertilization, and extracorporate development, provides a model of choice for transgenic research (Stuart et al., 1990, Culp et al., 1991, Hammerschmidt et al., 1999).
This model can be used, for example, to examine the impact of alteration of the Ikaros program of gene expression on definitive hematopoiesis in adults, within the short period of hematopoietic cell determination in ZF embryonic development.
As described herein, a transgenic Zebrafish (ZF) animal model provides an excellent model of vertebrate hematopoiesis.